The long term objective of this project is to elucidate the mechanism by which hormones, acting at the molecular level, regulate the synthesis of glucose in the mammalian liver. Clarification of these mechanisms will provide basic information that should improve understanding and management of disorders of carbohydrate metabolism in man such as diabetes mellitus, Cushing's syndrome, infantile hypoglycemias, and the hypoglycemia of toxic shock. A major goal of this period is to determine definitively the relative contributions that inhibition of the pyruvate kinase reaction and increased formation of phosphoenolpyruvate make to the overall stimulation of gluconeogenesis by glucagon and the "calcium-dependent" hormones, as typified by angiotensin II. Current estimates of these relative contributions vary widely and are dependent on the techniques employed. Rat hepatocyte preparations will be used for these studies, and the major techniques used to assess the relative importance of different control sites will employ isotopic tracers and the measurement of metabolic intermediates. Experiments are planned to resolve the conflicting data in the literature by comparing results obtained using different analytic methodologies within the same experiments. Variation of conditions including substrate concentrations and cytosolic pyridine nucleotide redox potentials, will be used to define the limitations and range of usefulness of each method. A second major goal is to understand the mechanism by which hormones produce acute changes in the concentrations of a number of intracellular compounds related to gluconeogenesis. In particular, the dramatic effects on glutamate and Alpha-ketoglutarate are of interest. The approach to this problem includes analysis of the sequence of rapid changes in metabolites in the cytosolic and mitochondrial compartments after hormone addition. Use of metabolic inhibitors, biotin-deficient rats, and variations of experimental conditions such as substrates and cytosolic pyridine nucleotide redox potential will assist in understanding the early events set in motion by hormones. A minor goal is to study the respiratory capacity of mitochondria within hepatocytes treated with gluconeogenic hormones. Emphasis will be on elimination of potential artifacts in these measurements. Another minor goal is to determine the effect of glucagon treatment on the degree of inhibition of the mitochondrial ATPase by its intrinsic peptide inhibitor.